Quantitative analysis of urinary organic acid]

We showed the optimum conditions for analysis of urinary organic acids using solvent extraction and GC/MS by the stable isotope dilution technique. Examples of the diagnosis in a case of heterozygote of ornithine transcarbamylase deficiency and a case of multiple carboxylase deficiency were described. The accurate quantitative analysis of urinary organic acids with stable isotope dilution technique is necessary for routine examination conducted in clinical laboratories.     

Detection of ornithine transcarbamylase deficiency heterozygotes by measuring of urinary uracil

The importance of detecting heterozygosity for X-linked ornithine transcarbamylase deficiency is well known. Although the DNA analysis and the allopurinol loading tests are commonly used for this purpose, both methods require complicated procedures. In order to establish a simple test for detecting female heterozygotes, we examined the uracil and orotic acid in single-voided urine samples from 70 healthy women, and from 12 asymptomatic females with ornithine transcarbamylase deficiency. Based on the results of healthy women, we were able to determine a screening cut-off line of 11.9 micromol/mmol creatinine (mean +/- 1SD in logarithmic form) for uracil. Using this cut-off line, the sensitivity of OCT heterozygotes was 100%. We were also able to establish a second cut-off line of 28.9 micromol/mmol creatinine (mean +/- 3SD in logarithmic form) for diagnosis. Using this second cut-off line, the specificity of OCT heterozygotes was 100%. Our study has shown that the measurement of urinary uracil is a relatively simple and effective method for detecting female heterozygotes.     

Determination of urinary orotic acid and uracil by capillary zone electrophoresis

We describe a simple method for measuring orotic acid and uracil concentration in urine by capillary zone electrophoresis in 20 mM Na-borate buffer, pH 9.2. The method was applied for studying a patient with HHH (hyperomithinemia, hyperammonemia and homocitrullinuria) syndrome. A high value of uracil excretion was found during periods of relatively low orotic acid excretion and normal ammonemia. The orotic acid level in urine was increased by increasing protein intake.     

醛固酮合成酶基因-344 T/C多态性与高血压病早期肾损害的关联研究

目的研究醛固酮合成酶基因-344T／C多态性是否与汉族人原发性高血压患者早期肾损害关联。方法放射免疫法检测225例1、2级原发性高血压患者24h尿白蛋白含量和血浆醛固酮浓度，24h尿白蛋白〈30mg判为正常白蛋白尿组，24h尿白蛋白在30～300mg为微量白蛋白尿组；聚合酶链反应．限制性片段长度多态性技术检测225例高血压患者和135名正常对照醛固酮合成酶基因-344T／C多态性。结果对照组、原发性高血压白蛋白尿正常组和微量白蛋白尿组之间TT、TC和CC3种基因型分布差异无统计学意义（P〉0．05）；3组之间比较T、C等位基因频率分布差异有统计学意义，与对照组、原发性高血压白蛋白尿正常组比较，微量白蛋白尿组C等位基因频率显著增高（P〈0．05）。与非C等位基因携带者相比较，C等位基因携带者血浆醛固酮浓度显著升高，24h尿白蛋白排泄量显著增加（P〈0．05）。结论醛固酮合成酶基因-344T／C多态性与汉族人原发性高血压患者早期肾损害关联，C等位基因可能是原发性高血压患者合并早期肾损害的遗传易感因子。     

The Effect of Two Methods of Pomegranate (Punica granatum L) Juice Extraction on Quality During Storage at $4^\circ$ C

The effect of two extraction methods of pomegranate juice on itsquality and stability was evaluated. The first method consistedof separation of the seeds from fruits and centrifugation. Thesecond method consisted of squeezing fruit halves with anelectric lemon squeezer. During a period of 72 hours of coldstorage at 4^°C, the juices were evaluated for thepresence of sugars, organic acids, and anthocyanins. Delphinidin3-glucoside was identified to be the major anthocyanin present atthe level of 45–69mg/L. Among the organic acids, oxalic andtartaric acids dominated. The major sugars detected inpomegranate juice were glucose and sucrose. No significantdifferences in the content of sugars, organic acids, oranthocyanins in juices obtained through application of the twodifferent extraction methods were detected, with the exception ofthe drastic decrease of cyanidin 3,5-diglucoside level in juiceobtained by seed centrifugation. The pH did not show differencesbetween treatments. Titrable acidity and the level of sugarsexpressed as ^°Brix decreased after 32 and 15 hoursafter extraction, respectively, when juice was obtained bycentrifuging the seeds.     

Diagnosis of inborn errors of metabolism using filter paper urine, urease treatment, isotope dilution and gas chromatography-mass spectrometry

This review will be concerned primarily with a practical yet comprehensive diagnostic procedure for the diagnosis or even mass screening of a variety of metabolic disorders. This rapid, highly sensitive procedure offers possibilities for clinical chemistry laboratories to extend their diagnostic capacity to new areas of metabolic disorders. The diagnostic procedure consists of the use of urine or filter paper urine, preincubation of urine with urease, stable isotope dilution, and gas chromatography-mass spectrometry. Sample preparation from urine or filter paper urine, creatinine determination, stable isotope-labeled compounds used, and GC-MS measurement conditions are described. Not only organic acids or polar ones but also amino acids, sugars, polyols, purines, pyrimidines and other compounds are simultaneously analyzed and quantified. In this review, a pilot study for screening of 22 target diseases in newborns we are conducting in Japan is described. A neonate with presymptomatic propionic acidemia was detected among 10,000 neonates in the pilot study. The metabolic profiles of patients with ornithine carbamoyl transferase deficiency, fructose-1,6-bisphosphatase deficiency or succinic semialdehyde dehydrogenase deficiency obtained by this method are presented as examples. They were compared to those obtained by the conventional solvent extraction methods or by the tandem mass spectrometric method currently done with dried filter blood spots. The highly sensitive, specific and comprehensive features of our procedure are also demonstrated by its use in establishing the chemical diagnosis of pyrimidine degradation defects in order to prevent side effects of pyrimidine analogs such as 5-flurouracil, and the differential diagnosis of three types of homocystinuria, orotic aciduria, uraciluria and other urea cycle disorders. Evaluation of the effects of liver transplantation or nutritional conditions such as folate deficiency in patients with inborn errors of metabolism is also described.     

Fetal fuels. V. Ketone bodies inhibit pyrimidine biosynthesis in fetal rat brain

Ketonemic states complicating late pregnancy are accompanied by lower brain weights in the newborn. Potential mechanisms whereby ketone bodies might inhibit cell proliferation were therefore examined in the fetal rat brain slice by measuring their impact on the de novo pathway for pyrimidine biosynthesis. DL-beta-hydroxybutyrate (10.8 mM) and acetoacetate (5.4 mM) were both found to diminish the incorporation of NaH14CO3 into [14C]UMP by 30%. This effect was similar in fetal tissues from fed and 48-h starved mothers. Graded concentrations of DL-beta-hydroxybutyrate (1.4-43.2 mM) resulted in a progressive inhibition that could not be explained either by isotope dilution consequent to ketone body oxidation or by a generalized inhibition of protein synthesis. The inhibition was not reversed with 10 mM glutamine, the principal nitrogen substrate for de novo biosynthesis of pyrimidines. When the conversion of orotic acid into UMP was blocked with 6-azauridine, DL-beta-hydroxybutyrate (10.8 mM) inhibited the incorporation of NaH14CO3 into orotic acid by 28%. By contrast, maximally inhibitory concentrations of this ketone body (43.2 mM) had no effect on the incorporation of [6-14C]orotic acid into [14C]UMP. Is is concluded that ketone bodies inhibit the de novo biosynthesis of pyrimidines in fetal brain slices and that they do so at a site proximal to orotic acid formation.     

DN患者血清HGF的表达及其临床意义

目的：探讨血清肝细胞生长因子（HGF）在DM2不同肾损害期中的变化及临床意义。方法：分别收集DM2患者105例,其中正常白蛋白尿组35例,微量白蛋白尿组32例,大量白蛋白尿组38例;糖耐量受损患者42例,采用ELISA检测血清HGF水平。患者同时于当日留取24h尿液,采用散射比浊法检测尿微量白蛋白。结果：糖耐量受损组和正常白蛋白尿组HGF浓度明显高于对照组（P〈0.05）;微量白蛋白尿组及大量白蛋白尿组HGF浓度明显低于对照组（P〈0.05）,且大量白蛋白尿组明显低于微量白蛋白尿组（P〈0.05）。结论：DM2患者不同肾损害期HGF浓度差别有统计学意义,检测血清HGF水平可为DN的诊断和治疗监测提供理论依据。     

Decreased fluorouracil cytotoxic effect on EB-virus transformed lymphocytes from hereditary orotic aciduria

5-Fluorouracil (5-FU), used widely for malignancies, phosphorylate mostly by uracil phosphoribosyl transferase (UPRT). Patients with hereditary orotic aciduria lack the orotate phosphoribosyl transferase (OPRT) activity. In the cancer cells, the OPRT activity is paralleled with the UPRT activity. This study shows that the UPRT activity of the hereditary orotic aciduria homozygote decreased about 40% of normal controls. Moreover, we investigated the 5-FU cytotoxic effects on hereditary orotic aciduria (one homozygote, 4 heterozygotes and 7 normal controls), using EB-virus transformed lymphocytes (EB-LC). 5-FU was addded to the culture medium at concentrations ranging from 0 to 10.0 micromol/l. The 5-FU cytotoxic effects on the homozygote were milder than those on controls at each 5-FU concentration. The 5-FU cytotoxic effects in the heterozygotes were at intermediate levels between the homozygote and controls. We speculate that 5-FU cytotoxic effects, both anti-tumor effects and adverse reactions, would be weak when a patient with hereditary orotic aciduria was treated with 5-FU.     

Renal effects of chronic exposure to organic solvents. A clinical controlled trial

Chronic effects of organic solvents on renal function were measured by creatinine clearances and urinary excretion rates of beta 2-microglobulin and albumin. Forty-three male printing trade workers occupationally exposed to different organic solvents for 9-25 years were compared with 43 age-matched male controls. No differences were found either in creatinine clearances or average basal levels of beta 2-microglobulin and albumin excretion rates, whereas a positive relation could be demonstrated between alcohol consumption on the day before the trial and urinary excretion rate of albumin. This investigation did not reveal any adverse renal effects of moderate chronic exposure to organic solvents in a group of active trade workers.     

Metabolic changes associated with hyperammonemia in patients with propionic acidemia

Propionic acidemia is an autosomal recessive disorder caused by deficiency of propionyl CoA carboxylase. Affected patients can develop severe hyperammonemia, whose causative mechanism is unknown. In this study, we monitored changes in metabolic parameters associated with hyperammonemia in patients with propionic acidemia. Levels of ammonia were correlated with plasma levels of individual amino acids and carnitine and with urinary organic acids. Significance of correlations was determined with analysis of variance. Hyperammonemia positively correlated with an increase in branched-chain amino acids (leucine and isoleucine) and a decrease in glutamine/glutamate and esterified carnitine. The urinary excretion of methylcitric acid, formed by the combination of propionic acid with oxaloacetate from the Krebs cycle, increased while that of citric acid decreased with hyperammonemia. These results suggest that in propionic acidemia, hyperammonemia is triggered by catabolism with the accumulation of propionic acid derivatives. The decrease of the plasma levels of glutamine/glutamate with hyperammonemia in patients with propionic acidemia indicates that the mechanism producing hyperammonemia differs from that in urea cycle defects. The increase in methylcitric acid and decline in citric acid urinary excretion suggest that hyperammonemia in propionic acidemia might be related to inability to maintain adequate levels of glutamine precursors through a dysfunctional Krebs cycle.     

Danish general practitioners'' estimation of urinary albumin concentration in the detection of proteinuria and microalbuminuria.

BACKGROUND. Microalbuminuria may predict proteinuria and increased mortality in non-insulin dependent diabetic patients. Early detection of microalbuminuria may therefore be essential. AIM. The primary objective of this study was to describe the association between the presence of albuminuria in diabetic patients as detected by general practitioners using conventional reagent strip dipstick tests for albumin, and the urinary albumin concentration as measured in a hospital laboratory. METHOD. A total of 675 newly diagnosed diabetic patients aged 40 years or over were included in the Danish study, diabetes care in general practice. Data for urinary albumin concentration from a morning urine sample and the results of three consecutive dipstick tests for albumin were collected for 417 patients. RESULTS. When defining elevated urinary albumin concentration as 200 mg l-1 or more (proteinuria) the finding of at least one positive test out of the three dipstick tests for albumin had a diagnostic sensitivity of 73% and a specificity of 89%. When the microalbuminuric range (15.0 to 199.9 mg l-1) was added to the definition of renal involvement, the sensitivity of the dipstick test became as low as 28% with a specificity of 96%. CONCLUSION. It is essential for general practitioners to be able to identify proteinuric patients. To achieve this by means of the conventional dipstick test, general practice procedures need to be improved. As it is becoming increasingly well-documented that microalbuminuric non-insulin dependent diabetic patients may benefit from pharmacological treatment of even slight arterial hypertension and heart failure, it seems reasonable to suggest that the use of dipsticks for albumin in general practice be replaced by laboratory quantitative determination of urinary albumin concentration in a morning urine sample.     

血清半胱氨酸蛋白酶抑制剂C和同型半胱氨酸联合检测在儿童早期糖尿病肾病中的诊断意义

目的探讨血清半胱氨酸蛋白酶抑制剂C（Cys-C）和同型半胱氨酸（HCY）联合检测对儿童糖尿病早期肾损伤的诊断意义。方法将92例糖尿病患儿根据24h尿白蛋白排泄率（UAER）分为3组（B、C、D）,其中B组为正常白蛋白尿组（UAER〈30mg/24h,33例）,C组为微量白蛋白尿组（30mg/24h≤UAER≤300mg/24h,28例）,D组为大量白蛋白尿组（UAER〉300mg/24h,31例）。选择同期42例正常体检儿童为对照组（A组）。同时测定各组儿童血清胱抑素C（Cys-C）、同型半胱氨酸（HCY）、肌酐（Scr）、尿素氮（BUN）和尿酸（UA）水平,采用Cockroft-Gault公式计算内生肌酐清除率（Ccr）,并对各组上述指标进行统计学比对分析。结果糖尿病各组患儿血清Cys-C、HCY、Scr、BUN、UA与对照组比较均升高,同时其水平随UAER的逐渐升高而升高,Ccr与对照组比较则逐渐下降。B、C、D3组中Cys-C、HCY水平与A组比较差异均有统计学意义（P〈0.05）。糖尿病各组各项指标两两比较差异均有统计学意义（P〈0.05）。血清Cys-C和HCY联合检测的阳性率为76.1%,高于Cys-C和HCY单独检测的阳性率（59.8%、56.5%,P〈0.05）。结论血清中Cys-C和HCY是反映早期肾功能损害的灵敏指标,Cys-C和HCY的联合检测可早期反映糖尿病肾损伤的程度。     

Pitfalls in measuring urinary proteins: age-related changes in urinary creatinine excretion that affect the urine protein/creatinine ratio

Knowing the amount of protein excreted in the urine is important in determining the severity and activity of renal diseases. In general, screening tests have been carried out using the urine dipstick. However, there are limitations in determining the amount of urinary protein excretion using qualitative tests for protein in spot urine samples due to the concentration and dilution of urine. Therefore, when using spot urine samples, it is helpful to calculate the urine protein/creatinine ratio (P/C) by simultaneous measurement of urinary creatinine for determining daily protein excretion. We examined P/C measurements using the dipstick method in 22,718 subjects who visited our hospital for health examinations. The results showed positive rates for qualitative urinary protein (1 + and more) of 4.2% for males and 2.7% for females. Also positive rates for P/C (150 mg/g.cre and more) were found of 7.7% for males and 10.2% for females. The results showed a reversal of positive rates for males and females compared with the results of qualitative urinary protein. In addition, P/C showed a higher positive rate in 70 years old or older both for males and females. The distribution of urinary creatinine levels simultaneously measured by dipstick method showed that the percentage of diluted urine with urinary creatinine level less than 50 mg/dL was 6.8% for males and 18.3% for females overall. Females showed a higher rate and the percentage tended to increase with age both for males and females. From these results, it was suggested that changes in urinary creatinine excretion with age that affect the P/C ratio are large. We then measured the albumin excretion rate in the 24-hour urine as well as examined the correlation between the urinary creatinine concentration and albumin index with regard to age and sex in 1,280 diabetic patients. The results showed that daily urinary creatinine excretion overall in males, overall in females, in males over 80 years old and in females over 80 years old were 1.063 +/- 0.428 g/day (mean +/- S.D.), 0.714 +/- 0.270 g/day, 0.666 +/- 0.201 g/day, and 0.531 +/- 0.017 g/day, respectively, which showed large variations. From these results, creatinine correction, in which uniformity of daily urinary creatinine excretion is the principle, is useful for assessment of short-term fluctuations in the individuals. However, it is indicated that there is a problem when used for screening, from young people to the elderly.     

Xanthine oxidase and uric acid as independent predictors of albuminuria in patients with diabetes mellitus type 2

Xanthine oxidase (XO) is an important enzyme responsible for conversion of purine bases to uric acid and represents the major source of reactive oxygen species (ROS) production in circulation. Since pathophysiological mechanism of the relationship between XO activity and urinary albumin excretion (UAE) rate is not well elucidated, we aimed to investigate this association in patients with diabetes mellitus type 2 (DM2). In addition, we wanted to examine whether uric acid itself plays an independent role in albuminuria onset and progression, or it is only mediated through XO activity. A total of 83 patients with DM2 (of them 56.6% females) were included in this cross-sectional study. Anthropometric, biochemical parameters and blood pressure were obtained. Multivariate logistic regression analysis showed that uric acid and XO were the independent predictors for albuminuria onset in patients with DM2 [odds ratio (OR) 1.015, 95% CI (1.008–1.028), p = 0.026 and OR 1.015, 95% CI (1.006–1.026), p = 0.040, respectively]. Rise in uric acid for 1 µmol/L enhanced the probability for albuminuria by 1.5%. Also, elevation in XO activity for 1 U/L increased the probability for albuminuria for 1.5%. A total of 66.7% of variation in UAE could be explained with this Model. Both XO and uric acid are independently associated with albuminuria in diabetes. Better understanding of pathophysiological relationship between oxidative stress and albuminuria could lead to discoveries of best pharmacological treatment of XO- and/or uric acid-induced ROS, in order to prevent albuminuria onset and progression.     

Quantitative determination of trimethylamine in urine by solid-phase microextraction and gas chromatography-mass spectrometry

Trimethylaminuria (fish odour syndrome) is diagnosed from an increase in urinary excretion of trimethylamine with decreased trimethylamine oxide. We report a new quantitative stable isotope dilution gas chromatography-mass spectrometry procedure for the analysis of these metabolites using solid-phase microextraction (SPME). Both polydimethylsiloxane and mixed Carboxen-polydimethylsiloxane SPME fibres were found to be suitable for the headspace extraction of TMA. This new sampling technique could have wide application for the analysis of volatile and semi-volatile compounds by metabolic screening laboratories.     

Purine and pyrimidine metabolizing activities in Trichomonas vaginalis extracts

Sixty-one purine and pyrimidine metabolizing activities were assayed in extracts of Trichomonas vaginalis. Of these, 43 were detected and quantitated. The only phosphoribosyltransfer activity observed was with uracil. No such activity was observed with adenine, guanine, hypoxanthine, xanthine or orotic acid. The rate of nucleoside cleavage was increased dramatically by the addition of inorganic phosphate. In addition, the extracts could catalyze the synthesis of ribonucleosides from the bases adenine, hypoxanthine, guanine and uracil but not cytosine, thymine or orotic acid, in the presence of ribose 1-phosphate. These data suggest that T. vaginalis contains primarily nucleoside phosphorylases instead of nucleoside hydrolases. Adenosine, deoxyadenosine, guanosine, deoxyguanosine, inosine, uridine, thymidine and GMP were phosphorylated in the presence of ATP. No nucleoside phosphotransferase activity was detected. Deamination of guanine, adenosine, deoxyadenosine, cytidine and deoxycytidine but not adenine was observed. These data suggest that salvage of adenine and guanine for ribonucleotide synthesis in T. vaginalis occurs via a phosphorylase/kinase pathway instead of through a phosphoribosyltransferase pathway which predominates in mammalian cells. In contrast, the pyrimidine base uracil can be converted to UMP via both a phosphoribosyltransferase or a phosphorylase/kinase pathway, analogous to that in mammals.     

Diseases of the kidney in children and adolescents with insulin-dependent diabetes mellitus

In order to estimate kidney functions in 61 juvenile diabetes patients, the glomerular filtration rate, urinary extraction of albumin during night, and systolic and diastolic artery blood pressures were examined. The ratio of these variables in relation to the duration of the disease, the metabolic control of the disease, age, and the interrelation of these elements were analysed. The control group consisted of twenty healthy children. The glomerular filtration rate was increased in 59% of patients. It shows a positive correlation with the duration of the disease and the increased urinary extraction of albumin. Tolerable values were established in relation to the systolic artery blood pressure. Diabetic incipient nephropathy (microalbuminuria) marks the increased urinary extraction of albumin. The prevalence of microalbuminuria in the group of children with diabetes was 27.86%. It was significantly higher in patients with a longer duration of the disease, in older patients and in those with an increased glomerular filtration rate. Microalbuminuria shows a positive correlation with glycogenic hemoglobin and systolic and diastolic artery blood pressure values. Systolic and diastolic artery blood pressures correlate with the duration of the disease and the increased urinary extraction of albumin. The results suggest that glomerular hyperfiltration is the most precise indication of disturbed renal functions. The high percentage of its occurrence is found when the disease was diagnosed and these adverse manifestations are highly reversible. The increased urinary extraction of albumin indicates structural renal lesions. The results also indicate the tendency of an increase of artery blood pressure, especially the diastolic pressure, ten years after the occurrence of the disease. High blood pressure accelerates the progress of diabetic nephropathy. The authors are of the opinion that the examination of kidney functions both in children and adolescents with insulin-dependent diabetes is necessary for the early detection of renal lesions and for the application of an adequate therapy.     

Adhesion/decalcification mechanisms of acid interactions with human hard tissues.

In order to study adhesion/decalcification mechanisms of acid interactions with human hard tissues such as bones and teeth, the chemical interaction of five carboxylic acids (acetic, citric, lactic, maleic, and oxalic) and two inorganic acids (hydrochloric and nitric) with enamel and two synthetic hydroxyapatite (HAp) powders with, respectively, a high and a low crystallinity were analyzed using X-ray photoelectron spectroscopy (XPS), atomic absorption spectrophotometry (AAS), and spectrophotometry (S). X-ray diffraction revealed that the crystallinity of the highly crystallized HAp was considerably higher than that of enamel while the crystallinity of the poorly crystallized HAp was similar to that of dentin and bone. XPS of acid-treated enamel demonstrated for all carboxylic acids ionic bonding to calcium of HAp. AAS and S showed for both HAps that all carboxylic and inorganic acids except oxalic acid extracted Ca significantly more than P, leading to a Ca/P ratio close to that of synthetic HAp (2.16 w/w). Oxalic acid extracted hardly any Ca, but substantially more P, leading to a significantly smaller Ca/P ratio than that of HAp. AAS showed that the calcium salt of oxalic acid hardly could be dissolved, whereas the calcium salts of all the other acids were very soluble in their respective acid solution. These results confirm the adhesion/decalcification concept (AD-concept) previously advanced. Depending on the dissolution rate of the respective calcium salts, acids either adhere to or decalcify apatitic substrates. It is concluded that the AD-concept that originally dictated the interaction of carboxylic acids with human hard tissues can be extended to inorganic acids, such as hydrochloric and nitric acid. Furthermore, HAp crystallinity was found not to affect the adhesion/decalcification behavior of acids when interacting with apatitic substrates, so that the AD-concept can be applied to all human hard tissues with varying HAp crystallinity.     

A new method for measuring urinary orotic acid

A relatively simple and specific method for measuring urinary orotic acid is reported. With this method, orotic acid is enzymatically converted to uridine 5-phosphate. The difference in absorbance produced by a simple colorimetric procedure in the presence and absence of the enzymatic conversion allows specific measurement of orotic acid. Normal values for urine are less than 4 micrograms/mg creatinine in infants and less than 3 micrograms/mg creatinine in older children and adults.